P382: PTK2B MEDIATES TYROSINE KINASE INHIBITOR DRUG RESISTANCE IN AML ASSOCIATED WITH ALTERED MIGRATION AND ADHESION PROPERTIES
Authors: C. Allert, S. Zimmermann, S. Göllner, D. Heid, M. Bruckmann, M. Janssen, B. Besenbeck, J. Krijgsveld, C. Müller-Tidow, M. F. Blank
Journal: HemaSphere (2022)

Background: Tyrosine kinase inhibitor (TKI) therapy is a well-established therapy approach in FLT3-mutated AML. However, TKI resistance occurs frequently while the underlying mechanisms remain incompletely understood. One concept of chemotherapy-resistance in leukemia is cell adhesion mediated drug resistance (CAM-DR). Aims: In this study we aimed to investigate the role of CAM-DR at an early stage of therapy resistance in FLT3-ITD mutated AML. Methods: We characterized FLT3-ITD mutated AML cell lines upon shorter and longer TKI exposure. Emerging TKI resistance was assessed by transcriptome and total proteome analyses, as well as nascent proteomics and MS-based protein stability/degradation assays. Further, we investigated the impact of emerging TKI-resistance on cell adhesion and migration. After identification of a highly expressed protein in TKI resistant cells with implications in cell adhesion, i.e. PTK2B, we investigated the effects of available inhibitors of this protein on AML cell lines in the presence or absence of other TKIs or chemotherapeutic agents and confirmed the effects in primary AML patient samples (n=6). Results: We discovered that protein tyrosine kinase 2b (PTK2B) was induced during early TKI resistance development. Interestingly, PTK2B, a member of the focal adhesion kinase superfamily, was upregulated at the transcriptional and translational level. PTK2B was also post-translationally stabilized in TKI resistant cells. Of note, PTK2B was highly expressed in leukemic stem cells (LSCs) from FLT3-ITD mutated patients. The upregulation of PTK2B was accompanied by increased migration of TKI-resistant cells with concomitant loss of cell adhesion, suggesting altered niche interactions. Notably, treatment with a PTK2B inhibitor, such as PF-431396, reverted the resistance-associated phenotypes. Enhanced migration was abolished upon PF-431396 treatment. Significantly altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon treatment of resistant cells with a PTK2B inhibitor. The FAK/PTK2B inhibitors PF-431396 and Defactinib synergized with several TKIs, such as midostaurin or gilteritinib, and commonly used chemotherapeutic agents, such as daunorubicin, in FLT3-ITD mutated AML cells. Synergism was restricted to FLT3 mutated cells. This synergism was significantly more pronounced in TKI-resistant cells and the addition of a PTK2B inhibitor could overcome TKI resistance to various extent. Also in primary AML patient samples, Defactinib showed lower IC50s specifically for FLT3-ITD mutated patients. Summary/Conclusion: These data establish that FLT3-ITD AML resistance towards TKI depends on PTK2B which alters leukemia cell adhesion and migration. PTK2B inhibitors might be synergistic with TKI in FLT3-ITD AML therapy. PTK2B inhibition could thus prevent the outgrow of drug-resistant clones at an early stage and may improve the outcome of FLT3-ITD mutated AML.